Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity

Biochemistry ◽  
1992 ◽  
Vol 31 (30) ◽  
pp. 6917-6924 ◽  
Author(s):  
Kimberly Carter Minghetti ◽  
Visala Chepuri Goswitz ◽  
N. Elise Gabriel ◽  
John J. Hill ◽  
Carlos A. Barassi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Specific Activity ◽  
High Specific Activity ◽  
Terminal Oxidase ◽  
Cytochrome O

Large-scale production of citrate synthase from a cloned gene

1982 ◽  
Vol 60 (12) ◽  
pp. 1143-1147 ◽  
Author(s):  
Harry W. Duckworth ◽  
Alexander W. Bell
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Citrate Synthase ◽  
Specific Activity ◽  
Tetracycline Resistance ◽  
Scale Production ◽  
Ampicillin Resistance ◽  
Colicin E1 ◽  
Large Scale Production ◽  
Cloned Gene

Starting with a colicin E1 resistance recombinant plasmid which contains gltA, the gene for citrate synthase in Escherichia coli, we have constructed an ampicillin-resistance plasmid containing the gltA region as a 2.9-kilobase-pair insert in the tetracycline-resistance region of pBR322. Escherichia coli HB101 harbouring this plasmid, when grown on rich medium containing ampicillin, contains citrate synthase as about 8% of its soluble protein. The enzyme has been purified from this rich source and is identical to the chromosomal enzyme prepared previously in every property tested, except for specific activity, which is 64 U∙mg−1 as compared with 45–50 U∙mg−1 previously obtained. The N-terminal sequences of both enzymes are reported, and they are identical up to residue 16 at least. The overall yield of pure enzyme, starting with the cells grown in 15 L of medium, is 600–800 mg.

Preparation of Isotope-labeled Dihydroneopterin 3'-Triphosphate with High Specific Activity

Pteridines ◽  
1990 ◽  
Vol 2 (3) ◽  
pp. 169-174 ◽  
Author(s):  
Gerd Katzenmeier ◽  
Bruno Schwarzkopf ◽  
Quang Le Van ◽  
Cornelia Schmid ◽  
Adelbert Bacher
Keyword(s):  
Escherichia Coli ◽  
Adenylate Kinase ◽  
Specific Activity ◽  
High Specific Activity ◽  
Starting Material ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
Guanylate Kinase ◽  
Phosphoribosyl Transferase ◽  
Phosphoribosylpyrophosphate Synthetase

SummaryIsotope-labeled dihydroneopterin 3'-triphosphate with 3H at positions C-1' and C-2', respectively, has been prepared from isotope-labeled glucose as starting material. Glucose was first converted enzymatically to ribose 5-phosphate. GMP was subsequently obtained by the action of phosphoribosylpyrophosphate synthetase and guanosine phosphoribosyl transferase. It was subsequently phosphorylated to GTP in two steps using adenylate kinase and guanylate kinase. Dihydroneopterin triphosphate was prepared from GTP by the action of recombinant GTP-cyclohydrolase I from Escherichia coli. The method allows the incorporation of 3H and 14C isotope labels into any desired position of dihydroneopterin triphosphate. Rapid purfication procedures for phosphoribosylpyrophosphate synthetase and guanosine phosphoribosyl transferase as well as HPLC assays for their determinations are described.

Inhibition of Escherichia coli CTP synthase by glutamate γ-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis

2001 ◽  
Vol 356 (1) ◽  
pp. 223-232 ◽  
Author(s):  
Stephen L. BEARNE ◽  
Omid HEKMAT ◽  
Jennifer E. MacDONNELL
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Specific Activity ◽  
High Specific Activity ◽  
Competitive Inhibitor ◽  
Enzymic Activity ◽  
Open Chain ◽  
Tetrahedral Intermediate ◽  
Allosteric Effector ◽  
Ctp Synthase

Cytidine 5′-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate γ-semialdehyde. Glutamate γ-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate γ-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (Kis 0.16±0.03mM; Kii 0.4±0.1mM) and a competitive inhibitor with respect to ammonia (Ki 0.39±0.06mM) in the presence of GTP at pH8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate γ-semialdehyde. Although glutamate γ-semialdehyde exists in solution primarily in its cyclic form, Δ1-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Δ1-pyrroline-5-carboxylate (l-proline, l-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition. When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate γ-semialdehyde is decreased approx. 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis.

scholarly journals N-Acetyl-L-glutamate kinase from Escherichia coli: cloning of the gene, purification and crystallization of the recombinant enzyme and preliminary X-ray analysis of the free and ligand-bound forms

1999 ◽  
Vol 55 (7) ◽  
pp. 1350-1352 ◽  
Author(s):  
Fernando Gil ◽  
Santiago Ramón-Maiques ◽  
Alberto Marina ◽  
Ignacio Fita ◽  
Vicente Rubio
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
High Specific Activity ◽  
Unit Cell ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Space Group ◽  
Cell Parameters ◽  
E Coli ◽  
Carbamate Kinase

The gene for Escherichia coli N-acetyl-L-glutamate kinase (NAGK) was cloned in a plasmid and expressed in E. coli, allowing enzyme purification in three steps. NAGK exhibits high specific activity (1.1 µmol s−1 mg−1), lacks Met1 and forms dimers (shown by cross-linking). Crystals of unliganded NAGK diffract to 2 Å and belong to space group P6122 or its enantiomorph P6522 (unit-cell parameters a = b = 78.6, c = 278.0 Å) with two monomers in the asymmetric unit. Crystals of NAGK with acetylglutamate and the ATP analogue AMPPNP diffract to 1.8 Å and belong to space group C2221 (unit-cell parameters a = 60.0, b = 71.9, c = 107.4 Å), with one monomer in the asymmetric unit. NAGK crystallization will allow the determination of proposed structural similarities to carbamate kinase.

scholarly journals Platelet-derived growth factor. Isolation by a large-scale procedure and analysis of subunit composition

1981 ◽  
Vol 193 (3) ◽  
pp. 907-913 ◽  
Author(s):  
C H Heldin ◽  
B Westermark ◽  
A Wasteson
Keyword(s):  
Molecular Weight ◽  
Growth Factor ◽  
Gel Electrophoresis ◽  
Large Scale ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
High Specific Activity ◽  
Equilibrium Analysis ◽  
Platelet Derived Growth Factor ◽  
Sedimentation Equilibrium

Platelet-derived growth factor was purified from fresh platelets by a large-scale procedure not involving the use of SDS (sodium dodecyl sulphate). The product, 0.5 mg of platelet-derived growth factor, obtained from about 3 × 10(13) platelets migrated as a single component in analytical gel electrophoresis in the presence of SDS and showed no inhomogeneity on sedimentation-equilibrium analysis in the ultracentrifuge. It had a high specific activity, 2 ng of platelet-derived growth factor/ml (70pM) being equivalent to 1% (v/v) human serum in an assay for multiplication-stimulating activity. Amino acid analysis revealed that platelet-derived growth factor contains all the common amino acids, except tryptophan, but no hexosamine. The molecular weight of platelet-derived growth factor, as determined by sedimentation-equilibrium analysis, was about 33 000. A similar value was obtained by gel electrophoresis in SDS under non-reducing conditions. In the presence of reducing agents the factor molecule was converted into two distinct components of lower molecular weight (17 000 and 14 000 respectively), as demonstrated by protein staining. The molecular model implicated by these findings is that platelet-derived growth factor consists of two different polypeptides chains, linked by disulphide bridges.

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